A commonly encountered situation in genetic analysis entails the need to identify a low percent of variant DNA sequences (“target sequences”) in the presence of a large excess of non-variant sequences (“reference sequences”). Examples for such situations include: (a) identification and sequencing of a few mutated alleles in the presence of a large excess of normal alleles; (b) identification of a few methylated alleles in the presence of a large excess of unmethylated alleles (or vice versa) in epigenetic analysis; (c) detection of low levels of heteroplasmy in mitochondrial DNA; (d) detection of drug-resistant quasi-species in viral, bacterial or parasitic infections and (e) identification of tumor-circulating DNA in blood of cancer patients (where people are suspected of having cancer, to track the success of cancer treatment or to detect relapse) in the presence of a large excess of wild-type alleles.
COLD-PCR methods for enriching the concentration of low abundance alleles in a sample PCR reaction mixture were initially described in a published patent PCT application entitled “Enrichment of a Target Sequence”, International Application No. PCT/US2008/009248, now U.S. Ser. No. 12/671,295, by Gerassimos Makrigiorgos which is incorporated herein by reference. The described COLD-PCR enrichment methods are based on a modified nucleic acid amplification protocol which incubates the reaction mixture at a critical denaturing temperature “Tc”. The prior patent application discloses two formats of COLD-PCR, namely full COLD-PCR and fast COLD-PCR.
In full COLD-PCR, the reaction mixture is subjected to a first denaturation temperature (e.g., 94° C.) which is chosen to be well above the melting temperature for the reference (e.g., wild-type) and target (e.g., mutant) sequences similar to conventional PCR. Then, the mixture is cooled (e.g., to 70° C.) to facilitate the formation of reference-target heteroduplexes by hybridization. In the basic full COLD-PCR method, lowering of the temperature from the first denaturing temperature (e.g., 94° C.) to the hybridization temperature (e.g., 70° C.) over a relatively long time period (e.g., 8 minutes) or retaining the reaction mixture at the hybridization temperature for a relatively long time period (e.g., 70° C. for 8 min) is required to assure proper hybridization. Once cooled, the reaction mixture contains not only reference-target heteroduplexes but also reference-reference homoduplexes (and to a lesser extent target-target homoduplexes). When the target sequence and reference sequence cross hybridize, minor sequence differences of one or more single nucleotide mismatches or insertions or deletions anywhere along a short (e.g., <200 bp) double stranded DNA sequence will generate a small but predictable change in the melting temperature (Tm) for that sequence (Lipsky, R. H., et al. (2001) Clin Chem, 47, 635-644; Liew, M., et al. (2004) Clin Chem, 50, 1156-1164). Depending on the exact sequence context and position of the mismatch, melting temperature changes of 0.1-20° C., are contemplated. Full COLD-PCR, as described in the above referred patent application, is premised on the difference in melting temperature between the double stranded reference sequence and the hybridized reference-target heteroduplexes. After cooling down to form reference-target heteroduplexes, the reaction mixture is incubated at a critical denaturing temperature (Tc), which is chosen to be less than the melting temperature for the double stranded reference sequence and higher than the lower melting temperature of the reference-target heteroduplexes, thereby preferentially denaturing the cross hybridized target-reference heteroduplexes over the reference-reference homoduplexes.
The critical denaturing temperature (Tc) is a temperature below which PCR efficiency drops abruptly for the reference nucleic acid sequence (yet is sufficient to facilitate denaturation of the reference-target heteroduplexes). For example, a 167 bp p53 sequence amplifies well if the PCR denaturing temperature is set at 87° C., amplifies modestly at 86.5° C. and yields no detectable product if PCR denaturation is set at 86° C. or less. Therefore, in this example the selected Tc should be −86.5° C. or less. After intermediate incubation at the critical denaturing temperature (Tc), the primers are then annealed to the denatured target and reference strands from the denatured heteroduplexes (e.g., 55° C.) and extended by a polymerase (e.g., 70° C.), thus enriching the concentration of the target sequence relative to the reference sequence. One of the advantages of full COLD-PCR is that the same primer pair is used for both target and reference sequences.
The above described full COLD-PCR method requires significant cycle times to ensure suitable cross-hybridization of reference-target heteroduplexes and has also otherwise proven to be somewhat inefficient. To address these issues, Makrigiorgos has described the use of reference blocking sequences to improve the efficiency and reduce cycle time of full COLD-PCR, see Full COLD-PCR Enrichment with Reference Blocking Sequence, International Application No. PCT/US2011/027473, published as Publication No. WO2011/112534, filed on Mar. 8, 2011, which is also incorporated herein by reference. This modified, full COLD-PCR method using reference blocking sequences is referred to as “RBS full COLD-PCR” for purposes herein. In the RBS full COLD-PCR method, reference blocking sequence is added at an excess concentration level to the amplification reaction mixture. The reference blocking sequence is a nucleic acid sequence complementary with at least a portion of one of the strands of the reference sequence between its primer binding sites, or partly overlapping the primer binding sites. The reference blocking sequence added to the reaction mixture is desirably single stranded (but can also be double stranded inasmuch as the initial denaturing step will result in denatured, single stranded reference blocking sequences). The reaction mixture is subjected to a first denaturing temperature, e.g. 95° C., which is above the melting temperature (Tm) of the reference sequence and also the target sequence, and results in denatured strands of the reference sequence and the target sequence. The reaction mixture is cooled to promote hybridization, for example to about 70° C. Since the cooling down occurs in the presence of an excess amount of reference blocking sequences, the reference blocking sequences preferentially hybridize with the complementary strand of the reference sequence, and also the complementary strand of the target sequence. For example, assuming that single stranded reference blocking sequence is added in excess at the beginning of the process, the reaction mixture at this point in the process will contain heteroduplexes of the reference blocking sequences and the complementary reference (e.g., wild-type) strand and heteroduplexes of the reference blocking sequences and the target (e.g. mutant) strands. The reaction mixture at this point also contains the denatured negative strands for the reference and target sequences. The formed heteroduplexes present in the RBS full COLD-PCR cycle are fundamentally different from the reference-target heteroduplexes formed in the unmodified full COLD-PCR protocol. Supplying an excess amount of reference blocking sequence promotes faster hybridization (e.g., about 30 seconds) than in the unmodified full COLD-PCR protocol (e.g., about 8 minutes); and the cool down hybridization step in the RBS full COLD-PCR protocol is less than one minute in duration.
In the RBS full COLD-PCR method, the reaction mixture is then subjected to a critical temperature (e.g., Tc=84.5° C.) which is sufficient to permit preferential denaturation of the target strands from the reference blocking sequence. The melting temperature for the duplex of the reference blocking sequence and the target strands will always be less than the melting temperature of the duplex of the reference blocking sequence and the complementary reference strand because the former contains a mismatch whereas the latter does not. The critical temperature (Tc) is selected so that duplexes of the reference blocking strands and the complementary reference strands remain substantially undenatured when the reaction mixture is incubated at Tc yet duplexes of the reference blocking strands and the target strands substantially denature. The term “substantially” means at least 60%, and preferably at least 90% or more preferably at least 98% in a given denatured or undenatured form.
After preferential denaturation, the temperature of the reaction mixture is reduced (e.g. 55° C.) so as to permit the primer pairs to anneal to the free target and reference strands in the reaction mixture. Again, assuming that single stranded reference blocking oligonucleotides are added in excess at the beginning of the process, at this point in the cycle there are, theoretically, two free strands of the target sequence compared to the initial denaturation step and only one free reference strand. The other reference strand is hybridized with the reference blocking sequence, and is therefore unavailable for amplification. The annealed primers are then extended (e.g., 70° C.), thus resulting in exponential amplification of the target sequence, while the reference strand is only amplified linearly. Accordingly, the target sequence is gradually enriched relative to the reference sequence in the sample during the full COLD-PCR cycles. The above steps are likely repeated ten to thirty cycles or more.
The reference blocking sequence is desirably at least several bases smaller than the target and reference sequences, on each side of the sequence so that the primers do not bind appreciably to the reference sequence and so that the reference blocking sequence is not extended by the primers that amplify the target sequence. To this end, optionally the 3′ OH end of the reference blocking sequence can be blocked to DNA-polymerase extension. Also, optionally, the 5′-end of the reference blocking sequence may be designed such that the nucleotide sequence partially overlaps the primer binding sites such that 5′ to 3′ exonucleolysis by Taq DNA polymerases (i.e. degradation of the hybridized reference blocking sequence) may be prevented.
Fast COLD-PCR, as described in the above incorporated patent application, International Application No. PCT/US2008/009248, now U.S. Ser. No. 12/671,295, by Gerassimos Makrigiorgos, is premised on there being a difference in melting temperature between the double stranded reference sequence (e.g., wild-type sequence) and the double stranded target sequence (e.g., mutant sequence). In particular, the melting temperature of the target sequence must be lower than the reference sequence. The critical denaturing temperature (Tc) in fast COLD-PCR is a temperature at or below which PCR efficiency drops abruptly for the double stranded reference nucleic acid sequence, yet is still sufficient to facilitate denaturation of the double stranded target sequence. During the fast COLD-PCR enrichment cycle, the reaction mixture is not subjected to denaturation at a temperature (e.g., 94° C.) above the melting temperature of the reference sequence as in the first step of the full COLD-PCR cycle. Rather, the reaction mixture is incubated at a critical denaturing temperature (e.g., Tc=83.5° C.), which is chosen either (a) to be less than the melting temperature for the double stranded reference sequence and higher than the lower melting temperature of the double stranded target sequence, or; (b) to be lower than the Tm of both reference and target sequences, whilst still creating a differential between the degree of denaturation of reference and target sequences. After incubation at the critical denaturing temperature (Tc), the primers are annealed to the denatured target strands and extended by a polymerase, thus enriching the concentration of the target sequence relative to the reference sequence. Again, the same primer pair is used for both target and reference sequences.
The use of fast COLD-PCR is limited to applications in which the melting temperature of the double stranded target sequence is suitably less than the melting temperature for the double stranded reference sequence. For example, mutations will not be detectable in sequencing data for a sample with a low abundance of mutant sequences that has been subjected to fast COLD-PCR if the melting temperature of the mutant sequence is the same or higher than the melting temperature of the wild-type sequence.